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The aim of the project was to determine the sequence of the gene encoding glucose-6-phosphate dehydrogenase (G6PDH) in the industrially used, but genetically little explored yeast Candida utilis. In the first strategy, a C. utilis DNA library was constructed in the expression vector phage lambda gt11, and was screened immunologically with antiserum raised against C. utilis G6PDH. The second strategy involved construction of libraries in the plasmid vector pTZ18R and screening with a mixed oligonucleotide probe. Two methods were developed that allow DNA, of appropriate quality for the construction of DNA libraries, to be prepared from C. utilis cells. Both involve centrifugation of C. utilis lysates through caesium trifluoroacetate gradients. A rabbit antiserum was raised against a commercial preparation of C. utilis G6PDH. It reacted with Western blotted C. utilis G6PDH, and non-specific binding of the antiserum to Western blotted E. coli protein was reduced by treatment with an E. coli extract. Antiserum pretreated in this way was used for immunological screening. Sequence information was determined for the inserts of 7 phage identified during immunological screening. None of the insert sequences was considered to contain part of the C. utilis G6PDH coding sequence. One putative positive colony was identified during the screening of the pTZ18R libraries. Sequence determination showed the insert to contain a region complementary to 16 bases in one of the 17-base long probe oligonucleotides. This insert was considered not to contain part of the C. utilis G6PDH coding sequence. Time did not permit further work which, using the methods developed, should now permit determination of the C. utilis G6PDH gene sequence.

The homoprotocatechuate degradative pathway of Escherichia coli C contains two isomerisation reactions with chemically similar intermediates, which were thought to be catalysed by distinct but genetically linked enzymes. The possibility that these two isomerases may have arisen from the same ancestral precursor, given their similar substrate structures and close physical location of their genes, was investigated by nucleotide sequencing and purification of the individual enzymes. The purified proteins are of very different subunit molecular weight and have been shown by kinetic measurements to be specific for their respective substrates. The two isomerisation events are separated by an enzyme catalysed decarboxylation and it has been shown that the second isomerisation and the decarboxylation reactions are distinct activities of the same protein subunit. Comparison of the amino acid sequence of the latter with that of the first isomerase of the pathway, reveals a very low level of similarity, suggesting that the two enzymes are unlikely to be derived from a common ancestor. Subcloning of the rest of hpc gene cluster has revealed that the gene order and direction of transcription are not as previously reported. All the genes for the pathway enzymes are transcribed in the same direction and are subject to negative regulation by a protein which appears to be transcribed in the opposite direction. A putative operator site to which the regulatory protein could bind has been located in the same region as a mapped promoter for the pathway genes, which also contains a binding site for the catabolite activator protein. Pairwise comparison of the amino acid sequences of the rest of the Hpc enzymes have shown only low levels of similarity. However, the single dehydrogenase enzyme of the pathway is similar to isoforms of human aldehyde dehydrogenase. The subcloning procedures used in this study have enabled high level expression of several of the pathway enzymes. This has enabled preliminary crystallographic analysis of the isomerase and decarboxylase/isomerase enzymes.

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